The objective of this research is to devise and implement new strategies for the mechanism-based inactivation of enzymes that cleave phosphate ester bonds. These inhibitors will be used to map the active sites, and define the catalytic mechanisms of the enzymes described below. Specific aims for the next grant period include: (1) Synthesis and testing of mechanism-based inactivators of prostatic acid phosphatase. This project includes the (a) analysis of the mode of inactivation brought about by fluoromethyl phenyl phosphate which should inactivate the phosphatase through enzyme-catalyzed formation of a quinone methide at the active site, (b) synthesis and analysis of the mode of action of 1-haloenol phosphates, which should inactivate the enzyme through formation of ketene at the active site, (c) synthesis and analysis of the mode of action of 2-haloenol phosphates, which should inactivate the phosphatase by formation of a haloacetaldehyde at the active site. (2) Synthesis and testing of phospholipase D inhibitors based on strategies described above. (3) Synthesis and testing of ribonuclease A inhibitors based on the strategy described above. In addition, design elements for the synthesis of inhibitors of glucose 6-phosphatase, cAMP phosphodiesterase, phospholipase C, and phosphoserine/phosphothreonine phosphatases are also described.